Volker Matzeit

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single‐stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem‐loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling‐circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral‐strand DNA synthesis independently from its termination and also to identify the sequence within which rolling‐circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem‐loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem‐loop sequence. Here we show that viral‐strand DNA synthesis efficiently initiates at both sequences.

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.

The conserved nonanucleotide motif of the geminivirus stem-loop sequence promotes replicational release of virus molecules from redundant copies

Recombinant plasmids containing head-to-tail copies of different coat-protein replacement genomes of wheat dwarf virus (WDV) were used to study the mechanism leading to the release of replicating unit-length molecules in suspension culture cells of Triticum monococcum. For plasmids bearing two complete genomes, the viral unit bracketed by the two large intergenic regions (LIR) becomes preferentially released. Addition of a third copy of the LIR on the inoculum plasmid is necessary for release of both WDV genomes with the same efficiency. Using plasmids containing a single viral genome flanked by two different hybrid LIRs, we show that the sequence TAATATTA, which is part of the conserved geminivirus nonanucleotide motif of the potential hairpin structure, is the region of the LIR within which the release of unit-length molecules occurs. Moreover, the data suggest that this release results primarily from rolling-circle replication, also in situations where intramolecular homologous recombination is simultaneously possible.

DNA replication of wheat dwarf virus, a geminivirus, requires two cis-acting signals

Replication of the single-stranded DNA genome of wheat dwarf virus (WDV) leads to the accumulation of covalently closed double-stranded DNA of genome length in infected cells. By studying the replication properties of a naturally occurring deletion mutant of WDV isolated from infected plants and of deletion mutants constructed in vitro, we have defined cis-acting regions required for viral DNA replication. The results show that two distinct regions are required in cis to yield the normal replicative forms of WDV-DNA.

Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous plant cells.

Shuttle vectors have been constructed that are able to replicate in either Escherichia coli or plant cells. They contain the ColE1 origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants. Such plasmids are able to replicate in E. coli and wheat cells. The plasmids can be rescued in E. coli and show no changes during their passage through plant cells. Such an E. coli/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.

Fate and Expression of Vector DNA in Plant Cells

Plant transformation systems based on gene vectors derived from the Ti plasmid of Agrobacterium tumefaciens (16), on direct DNA uptake (6), and on wheat dwarf virus (WDV) DNA (14) were used to introduce a number of chimeric genes into, respectively, tobacco protoplasts, suspension culture cells of Triticum monococcum, and Zea mays. The actual genes involved in these studies were chosen to serve as models for investigating the fate, expression, recombination, and transposition of foreign DNA in plant cells.